The recurring chromosomal translocation (3;21)(q26;q22) has been associated with de novo or therapy related acute and chronic myeloid leukemias. This translocation is unusual in that it results in three different fusion genes. The role of each fusion gene is not known. One of them, AML1/MDS1/EVI1, results from the in-frame joining of two transcription activators following the translocation. They are AML1, which is necessary to fetal liver hematopoiesis and is located at chromosome band 21q22, and MDS1/EVI1, located at chromosome band 3q26. MDS1/EVI1 is not detected in hematopoietic tissues, but is transiently expressed in differentiating hematopoietic stem cells. In contrast to AML1 and MDS1/EVI1, which are very strong transcription activators, AML1/MDS1/EVI1 is a repressor, and can inhibit promoters activated by AML1 and MDS1/EVI1. The objective of this proposal is to dissect the molecular mechanisms by which the fusion protein AML1/MDS1/EVI1 alters the progress of the hematopoietic differentiation leading to leukemia. We have developed several models showing that, in hematopoietic precursor cells, AML1/MDS1/EVI1 inhibits the response to factors that control cell replication and differentiation. Based on preliminary results, we hypothesize that the fusion protein acts by disrupting the normal regulation of genes regulated by AML1 and MDS1/EVI1 during hematopoiesis, resulting in inappropriate level of expression of lineage- specific factors critical for hematopoietic differentiation and replication. The questions addressed in this proposal are: How is the repression mediated, and what are the targets of the fusion protein? By using a combination of biochemical and molecular techniques, and tissue culture studies, we will identify the target proteins regulated by AML1/MDS1/EVI1 and assess their role in cell transformation. The information we obtain will be useful in the design of new treatments for patients with myeloid leukemia and a t(3;21). In addition, our results will provide insight in the study of leukemias in which either AML 1 or MDS1/EVI1 are rearranged.